Despite the importance of mast cells (MCs) in innate and acquired immunity, their development and recruitment into the skin is not well understood. Our findings suggest that the skin microbiome has a strong influence on migrating and, in particular, maturing MCs in the skin. Based on our preliminary data, we have demonstrated that bacterial products at the skin surface participate in the regulation of MC antimicrobial functions, homing to the skin, and help them respond to their survival growth factor, SCF. Even more important is the ability of the microbiome to induce an anti-inflammatory phenotype on human MCs that contributes to maintaining skin homeostasis, which helps avoid unnecessary inflammation. Antibiotic exposure, and a pseudo-aseptic environment, have the potential to alter the skin microbiome selectively and may be the cause of the increasing incidence of human disorders such as atopic dermatitis. We hypothesize that bacterial products, increase mast cell recruitment and are responsible for maintaining high levels of antimicrobial peptides (AMPs) and a low pro-inflammatory cytokine profile. A humanized mouse model system will enable the simultaneous study of both human MC migration pathways and TLR innate immune responses in a genetically adaptable organism, thereby translating our findings to human skin. We propose the following: a) Determine whether bacterial products from the skin microbiome are required for normal mast cell localization and function. The use of germ free and antibiotic treated mice will allow us to test whether disruption of the normal skin microbiome results in improper localization and function in vivo and whether reconstituting the microbiome can reverse these effects. Because human MCs may have a different sensitivity to the microbiome, we will test our findings on human MCs (huMCs) to provide a foundation for future translational research. b) Clarify the signaling pathways that are responsible for MC sensing the skin microbiome and how activation of these pathways affects MC function. We will focus on characterizing the MC receptors involved in sensing bacterial byproducts. The aspects to be studied: 1. Commensal activity directly on MC receptors (TLR ligands), 2. Commensal activity on keratinocytes that generate ligands (SCF, IL-1, and S1P) for MC receptors, and 3. Commensal activity on Endothelial cells (SCF) that generate ligands for MC receptors. c) Determine how MC interaction with the microbiome modulates intracellular pathways in skin MCs and affects their function. Our preliminary data show that bacterial byproducts modulate huMCs maturation and activation through NF-kB, S1P and intracellular sphingokinases (Sphks) pathways to produce S1P; we have found that LTA and other commensal fractions induce the expression of specific miRNAs that interfere with TLR-NF-kB signaling.